A variety of analytical procedures have been developed for the separation and identification of different molecular species present in a specimen. Separation is generally accomplished by applying the specimen to a water-containing solid medium and inducing molecular separation of the species within the medium. In particular, chromatography and electrophoresis have been employed, both of which provide separation of different molecular species. The separation medium is generally called a chromatographic medium or electrophoretic plate. In such processes, a variety of reagents which interact with one or more of the molecular species in the specimen may also be applied to the medium before, during or after the separation process to assist in separation or identification of the analytes.
In general, poor resolution inhibits identification of separated materials in conventional electrophoretic media because of gross diffusion of the water-soluble reagents in the medium due to the presence of water. Proteins can be fixed to the medium with an acid or solvent to give quite sharp signals when stained. However, the fixing process usually destroys enzyme activity by denaturation. Further, the stains do not generally have the same affinity for all proteins to be determined.
Reagents have been introduced into the electrophoretic medium by a number of means. For example, U.S. Pat. No. 3,975,162 (issued Aug. 17, 1976 to Renn) describes the use of a transfer device composed of water-soluble reagents dispersed in a water-soluble binder. When the wetted device is placed on the surface of an electrophoretic plate, the binder dissolves and the water-soluble reagents diffuse into the plate to react with the separated analytes. This technique has a number of disadvantages. The electrophoretic plate can be used only once because the reagents diffuse in water and the resulting detectable signal is observed in the plate. Moreover, because reagent transfer is carried out in highly aqueous environment, resolution of the resulting analyte-reagent signals is poor. The reaction product (e.g. dyes) tends to spread out in the medium due to capillary action. Further, the procedure can be difficult to carry out.
U.S. Pat. No. 4,455,370 (issued June 19, 1984 to Bartelsman et al) describes the use of a microporous nylon membrane to transfer analytes, e.g. nucleic acids, from an electrophoretic plate to the membrane by electroblotting. All analytes are transferred since the membrane has limited selectivity. The membrane is then treated to provide a detectable signal in the presence of analytes. It does not contain reagents which provide a detection means. Further the transfer process is carried out in an aqueous environment.
Cellulosic membranes impregnated with water-soluble fluorogenic substrates have been described for use for identifying isoenzymes in an electrophoretic plate by R. E. Smith, in J. Histochem. Cytochem., 32(12), pp. 1265-1274 (1984). Smith claims that his membrane overlay provides highly resolved identification bands. However, these membranes have a number of serious drawbacks. The membrane overlay must be wet when used. As a result, the identification bands are quite diffuse because the substrates which provide a detectable signal are water-soluble. Further, the overlays do not provide a means for multiple testing with the same electrophoretic plate because substrate diffuses into the plate from the overlay. In essence, the Smith overlay can be used in a single test only.
The Smith overlay membranes are not transparent and are therefore limited in utility. They cannot be used for transmission analysis using colorimetric detection procedures.
It would be useful to have a means for overcoming the problems noted above. In particular, it would be desirable to have a means for detecting a plurality of electrically separated analytes using the same electrophoretic plate. It would also be useful to be able to use either colorimetric or fluorometric signals for detecting analytes.